The avidin-biotin affinity-based technology has found wide applicability in numerous fields of biology and biotechnology since the pioneering work by Dr. Edward Bayer and Dr. Meier Wilchek in the 1970""s. The affinity constant between avidin and biotin is remarkably high and is not significantly lessened when biotin in coupled to a wide variety of biomolecules. Further, this affinity is substantially maintained even when cerivatized forms of the biotin are employed and numerous chemistries have been identified for coupling biomolecules to biotin with minimal or negligible loss in the activity or other desired characteristics of the biomolecule. In certain applications, avidin is immobilized onto an inert material over which a solution containing biotinylated biomolecules is passed. The affinity of the biotin for the avidin provides for the separation of the biomolecule from the solution. A review of the biotin-avidin technology can be found in Applications of Avidin-Biotin Technology to Affinity-Based Separation, Bayer, et al., J. of Chromatography, 1990, pgs. 3-11.
EP 592242 describes a novel fibrin sealant based on fibrin monomer as opposed to the traditional fibrinogen-based sealants andinvolves subjecting fibrinogen to a thrombin-like enzyme which is preferably removed after such treatment. EP 592242 describes that the enzyme captureand removal can be accomplished by using biotinylated batroxobin which can be recaptured with an avidin material. This and other applications would benefit by more convenient forms of biotinylated biomolecule and avidin materials. Presently, these materials are sometimes difficult to work with, can be unstable, can lose enzyme activity in processing such as lyophilization, may be unduly hygroscopic and do not withstand sterilization processes.
In accordance with the present invention, novel compositions and methods for biotinylated biomolecules and the biotin/avidin affinity technology are described. The novel composition involving biotin comprises:
i) a biotinylated biomolecule;
ii) a biomolecule protectant;
iii) buffer means to maintain a desired pH; and
iv) one or more bulking agents selected from water soluble, nonionic polymers.
This composition is conveniently an aqueous solution and preferably includes an agent to protect the composition against instability during terminal sterilization. Most preferably, this composition is freeze-dried to provide a stable, irradiatable powder form of the biotinylated biomolecule. Methods for making a fibrin monomer material, useful, for example, in a fibrin sealant, are also disclosed.
The present invention discloses novel, stable compositions of biotin-biomolecule. The preferred compositions are freeze-dried and are stable, easy to handle, and can be terminally sterilized, e.g., by gamma irradiation without damage to the compositions. This is especially advantageous when the composition is a biotinylated biomolecule because it has been found to be very efficient to be able to terminally sterilize the lyophilized biotinylated biomolecule without damage to its activity. The present lyophilized biotin-based compositions have wide applicability wherever the avidin-biotin technology is useful because these compositions are water soluble, have low moisture uptake, have low bioburden, can be terminally sterilized (e.g., irradiated), remain stable and are pharmacologically acceptable. These advantages are provided by the unique combination of protectants and bulking agents as described herein.
These novel compositions include, along with the biotinylated biomolecule, a biomolecule protectant, buffer means to maintain the desired pH and one or more water soluble, nonionic polymer bulking agents. Preferably, the composition further includes an agent to protect the composition against deleterious effects of terminal sterilization, e.g., gamma irradiation. The biomolecule can be any desired enzyme or protein which is to be used in a biotinylated form. Numerous biotinylated biomolecules exist in the prior art and all of those prior biomolecules are useful herein as well. With regard to the novel fibrin monomer process in the above-referenced EP 592242, thrombin-like enzymes are useful in a biotinylated form. Such thrombin-like enzymes include thrombin or a thrombin-like enzyme selected from Acutin, Venzyme, Ancrod, Asperase, Batroxobin (from B. Altrox, B. Moojeni or B. Maranhao), Botropase, Crotolase, Flavoxogin and Gabonase. Nonlimiting examples of other biotinylated biomolecules include biotinylated lectins, antibodies, mitogens, DNA, RNA, tRNA, rRNA fragments, nucleosomes, membranes, membrane proteins, glycoproteins and synthetic peptides.
The biotin component of the biotinylated biomolecule can be biotin or any derivatized form or analog thereof, or any molecule having an affinity for avidin including monomeric avidin, Strept avidin, or any protein having biotin-binding properties including recombinant forms of any of the above. Patents and literature are replete with the various biotin compounds including various spacers, linking groups and the like, for use in the present applications. Nonlimiting examples can be found in M. D. Savage, et al. (1992), Pierce Chemical Co., Avidin-Biotin Chemistry: A Handbook; DE 3629194, U.S. Pat. Nos. 5,180,828, 4,709,037 and 5,252,743, 4,798,795, 4,794,082, WO 85/05638 incorporated herein by reference.
The biomolecule protectant of the novel biotin compositions is any agent capable of protecting the desired activity of the biomolecule and thereby imparting stability to the biomolecule composition. Biomolecule protectants include, but are not limited to, trehalose, glycerol, ammonium sulphate and amino acids. Preferably the biomolecule protectant is an amino acid and, more preferably, the amino acid is a simple zwitterion such as glycine, alanine and valine with glycine being most preferred.
The buffer means of the present biotin compositions can be any convenient buffer suitable for maintaining the pH of the composition at a desired level. In the fibrin monomer process of EP 592242 it is desired to maintain the biotinylated biomolecule at about pH7, therefore sodium barbital, citrate, sodium barbital phosphate, potassium phosphate, imidazole-HCI, piperazine, sodium bicarbonate-5% CO2, triethano amine-HCIxe2x80x94NaOH, tris (hydroxymethyl)aminomethane and sodium phosphate bufferare useful with sodium phosphate being preferred.
The bulking agent of the present biotin-biomolecule compositions is selected from water soluble, nonionic polymers. The bulking agent provides both chemical and physical stability to the present compositions and, for example, it presents the novel compositions when in the form of a freeze-dried cake from collapsing. The nonionic water soluble polymers also provide protection to the biomolecule. Dextran and similar polysaccharides have been found to enhance the stability of the present compositions. Nonlimiting examples of such bulking agents include dextran, polyvinylpyrrolidone, polyvinylalcohol, polyethyleneglycol, hydrolyzed starch and polysaccharides (e.g., lactose, glucose, maltose, mannitol, etc.) with dextran, especially dextrans having a molecular weight between 50,000 and 100,000 Daltons (e.g., Dextran T-70 from Pharmacia Co.) being preferred.
The optional terminal sterilization protectant is selected from antioxidants, free radical scavengers and reducing agents. Preferred are antioxidants such as reduced glutathione, xcex1-tocopherol, N,N-dimethyl-p-phenylenediamine and sodium ascorbate with sodium ascorbate being most preferred.
Preparation of the biotinylated molecule is accomplished by known techniques. For example, a biotin derivative (which can be any desired biotin compound with spacer arm and/or leaving groups as discussed above) such as N-hydroxysuccinimide-biotin (NHS-biotin) can be reacted with the desired biomolecule, e.g., the soluble enzyme Batroxobin, in a solvent and in the presence of a buffer. The NHS functions as a leaving group to provide the so-formed biotin Batroxobin. This can be purified using standard methodology, for example, subjecting the biotin Batroxobin to purification on a Sephadex(trademark) chromatography column to remove free biotin, NHS-biotin and other low molecular weight solutes.
Thereafter, an aqueous solution is prepared comprising the components of the composition, i.e., the biotinylated biomolecule, biomolecule protectant, buffer means and bulking agent. Preferably, the purification step above can utilize the buffer desired to be in the end product, which provides that water and bulking agent are added to the biotinylated biomolecule and buffer to form the aqueous solution.
The aqueous solution of this invention comprises:
0.01 to 50% by weight of biomolecule protectant;
1 to 50% by weight of bulking agent;
the biotinylated biomolecule in a concentration selected according to the particular application;
water; and
buffer necessary to maintain the desired pH.
This solution or suspension is also a useful, stable form of the biotinylated biomolecule and, as such, is considered a part of the present invention. If sterility is required, the solution can be prepared aseptically or can include the optional terminal sterilization protectant if terminal sterilization, e.g., gamma irradiation, is to be employed. The terminal sterilization protectant is typically present in the aqueous solution in an amount of from about 0.01% to about 10%.
These ranges are especially useful for compositions. containing from 0.1 to about 1.0 mg of enzyme or biomolecule per ml of composition and will also provide significant protection for compositions containing up to 5 mg of enzyme per ml of composition. Preferably, for compositions containing more than 1 mg/ml of enzyme and especially for compositions containing more than 5 mg/ml, the percentages of each component should be increased in a manner roughly proportional to the increase in enzyme concentration.
A preferred aqueous composition of the present invention comprises about 2% of biomolecule protectant, about 2% bulking agent, about 50 mM buffer,about 0.25% terminal sterilization protectant and the required concentration (preferably 0.1-0.5 mg/ml) of biomolecule.
Most preferred is when the biomolecule is Batroxobin in an amount of from about 50 to 200 activity units per milliliter of solution, and when the composition includes 2% by weight glycine, 50 millimolar sodium phosphate buffer (to maintain pH7), 2% by weight dextran and 0.25% by weight sodium ascorbate.
In a preferred embodiment of the present invention, the aqueous solution is lyophilized to provide a convenient powder composition typically in the, form of a cake. Lyphophilization techniques are well known and any suitable technique can be employed. One suitable lyophilization, i.e., freeze drying process involves pre-cooling the lyophilization apparatus to xe2x88x9245xc2x0 C., freezing the solution to xe2x88x9240xc2x0 C., warming the product to xe2x88x9225xc2x0 C. and holding for 11 hours or more, cooling the product to xe2x88x9243xc2x0 C., introducing a reduced pressure (i.e., vacuum) to about 0.1 millibar and maintaining reduced pressure at xe2x88x9243xc2x0 C. until drying is complete as is evidenced by cessation of water vapor evolution, reducing the pressure to the lowest setting while raising the temperature in 5xc2x0 C./hour increments to 30xc2x0 C. and holding the so-treated product at 30xc2x0 C. for at least 5 hours.
The compositions of this invention involving a biotinylated form of thrombin or a thrombin-like enzyme, e.g., Batroxobin, are useful to convert fibrinogen, or a fibrinogen-containing composition, into fibrin monomer, or a fibrin monomer-containing composition. Accordingly, the present invention further includes a novel method, to prepare a fibrin monomer useful, for example, in preparing a fibrin sealant. This novel method involves subjecting a source of fibrinogen to a stable, biotinylated thrombin or thrombin-like enzyme composition as defined herein to convert fibrinogen into fibrin monomer, xe2x80x9ccapturingxe2x80x9d the biotinylated enzyme with an avidin material, and removing the enzyme which is a part of the so-formed biotin/avidin complex.
Although in an ideal setting some of the avidin should xe2x80x9cleachxe2x80x9d from its agarose (or other inert) support and hopefully all of the biotinylated biomolecule is captured by the avidin/inert support material, it is understood that this may not always be the case. It has now been found that free avidin, leached from its inert support, is capable of coupling with a biotinylated biomolecule (e.g., baxtroxobin) or vice versa, affording capture and removal of the enzyme complex from solution. Accordingly, the reliability of the present compositions and methods are further enhanced by the self-scavenging medonium described herein.
The compositions of the present invention can further be incorporated into a processing unit, e.g., an automated centrifuge for preparing fibrin monomer as defined above. The biotinylated biomolecule composition can be preloaded into the processing unit in powder form or can be lyophilized in situ in the device or in a controlled release compartment of the device.
The biotinylation of the biomolecule can be accomplished, as discussed above, by any known biotinylation process. It has been found that careful control of the ratio of biotins to biomolecules is important in the ultimate desired performance of the biomolecule. For example, regarding biotinylated batroxobin for use in the process of preparing a fibrin monomer in EP 592242, it is important for the batroxobin to maintain sufficient activity so as to efficiently convert the fibrinogen to fibrin monomer. It is also important for the biotinylated batroxobin to be readily captured by the avidin material for thorough separation of the enzyme from the fibrin-monomer product. In the case of batroxobin, in theory, 14 biotin molecules can be coupled to the enzyme. In accordance with the present invention, it has been found that the mean number of biotin molecules per Batroxobin molecules in a composition should be in the range 5-12 and preferably 6-8. It is believed that if the mean is below about 5, that a significant number of Batroxobin molecules may actually not be biotinylated, resulting in incomplete enzyme capture. It has also been found that if the mean is above about 8, the batroxobin activity is reduced. This is believed to have applicability to other biomolecules as well, especially to the thrombin and thrombin-like enzymes. Accordingly, compositions containing biomolecules having 10 or more binding sites per biomolecule capable of reacting with a biqtinylation reagent should have a mean number of at least 5 and preferably 6 biotins/biomolecule. It should be understood by those skilled in the art that these preferred ranges of biotins per biomolecule can be reduced if any surface reaction sites on the biomolecule are hyperactive or if the biotinylation process involves physical protection of part of the biomolecule surface (from biotinylating agents), e.g., by reversibly binding the biomolecule to a solid surface.